Abstract

Epigenetic dysregulation has emerged as a recurring mechanism in the etiology of neurodevelopmental disorders. Two such disorders, CHARGE and Kabuki syndromes, result from loss of function mutations in chromodomain helicase DNA-binding protein 7 (CHD7LOF) and lysine (K) methyltransferase 2D (KMT2DLOF), respectively. Although these two syndromes are clinically distinct, there is significant phenotypic overlap. We therefore expected that epigenetically driven developmental pathways regulated by CHD7 and KMT2D would overlap and that DNA methylation (DNAm) alterations downstream of the mutations in these genes would identify common target genes, elucidating a mechanistic link between these two conditions, as well as specific target genes for each disorder. Genome-wide DNAm profiles in individuals with CHARGE and Kabuki syndromes with CHD7LOF or KMT2DLOF identified distinct sets of DNAm differences in each of the disorders, which were used to generate two unique, highly specific and sensitive DNAm signatures. These DNAm signatures were able to differentiate pathogenic mutations in these two genes from controls and from each other. Analysis of the DNAm targets in each gene-specific signature identified both common gene targets, including homeobox A5 (HOXA5), which could account for some of the clinical overlap in CHARGE and Kabuki syndromes, as well as distinct gene targets. Our findings demonstrate how characterization of the epigenome can contribute to our understanding of disease pathophysiology for epigenetic disorders, paving the way for explorations of novel therapeutics.

Specificity of the CHD7LOF and KMT2DLOF DNAm Classification SignaturesThe plot shows the predictions for all samples from the original Discovery Cohorts, as well as for 162 normal blood samples extracted from the GEO repository. The x axis shows the predictive scores generated from the CHD7LOF-specific predictive model derived using the CHD7LOF individuals and matching controls. The y axis shows the predictive score of the KMT2DLOF-specific predictive model derived using the KMT2DLOF individuals and matching controls. Importantly, using the KMT2DLOF-specific model all 19 CHD7LOF (red C) received low scores, along with all CHD7LOF matching controls (red circles) and all GEO samples (green crosses). Similarly, using the CHD7LOF-specific model all 11 KMT2DLOF (blue K) received low scores, along with all KMT2DLOF matching controls (blue diamonds) and all GEO samples (green crosses).

Validation of CHD7LOF and KMT2DLOF DNAm Classification Signatures on a Blinded CohortWe derived the scores for each sample using the two predictive models built for the CHD7LOF and KMT2DLOF DNAm classification signatures (x axis and y axis, respectively; see ), for a validation set of DNAm samples. This set included both pathogenic mutations and VUS in CHD7 (red squares), KMT2D (blue triangles) and KDM6A (turquoise diamond). The mutation and their pathogenicity were initially blinded and were revealed only after the prediction scores were determined. Importantly, all CHD7 mutations received low scores by the KMT2D-specific predictive model, and vice versa. Pathogenic mutations in CHD7 (filled red squares) received high scores from the CHD7LOF model, and pathogenic mutations in KMT2D (filled blue triangles) received very high scores from the KMT2DLOF model. Interestingly, a pathogenic mutation in the Kabuki-associated gene KDM6A also received a very high score from the KMT2DLOF model, indicating a potential methylation-signature overlap between these two genes.

Sequence Variants in CHD7 and KMT2D Sorted Using the CHD7LOF and KMT2DLOF DNAm Classification SignaturesWe derived the scores for each individual using the two models generated for CHD7LOF and KMT2DLOF DNAm classification signatures (x axis and y axis, respectively; see ), for a set of 13 mutation variants in CHD7 (red crossed circles) and 10 mutation variants in KMT2D (blue crossed squares). The details of the sample classification are shown in and .

Targeted Sodium Bisulfite Pyrosequencing Validation of DNAm Alterations in CHD7 and KMT2D Discovery Cohorts(A–C) DNAm was assessed for three CpG sites in the promoter of HOXA5 (cg01370449, cg04863892, and cg19759481). The gain of DNAm for the three sites in CHD7LOF: 18%, 20%, and 20%. For KMT2DLOF there was also a gain of DNAm: 18%, 18%, and 19%, respectively. Both the CHD7LOF and KMT2DLOFgroup are statistically different from the controls for all three probes, but not from each other.(D–F) DNAm was assessed for three CpG sites in the gene body of SLITRK5 (cg16787483, cg24626752, and cg09823859). A loss of DNAm of 20%, 14%, and 12% in the CHD7LOF samples and a gain of DNAm of 21%, 24%, and 24% in KMT2DLOF samples are shown. Both the CHD7LOF and KMT2DLOFgroup are statistically different from the controls for all three probes, and from each other.(G and H) DNAm was analyzed for FOXP2 (cg18546840 and cg18871253) in CHD7LOF, which had a 15% loss of DNAm compared to controls.(I) DNAm was analyzed for MYO1F (cg15254671) in KMT2DLOF, which had a loss of DNAm of 33% compared to controls. Testing for a statistical difference between all groups was performed using a Kruskal-Wallis test; ∗p < 0.0001.